The visceral organs are controlled and regulated by the autonomic nervous systems (ANS), which serves both visceral sensory and motor functions. End organ function is controlled via local reflex circuits governed by neurons located in the brainstem and spinal cord, suggesting that one approach to modify diseased end organ function is by modulation of the nerves that innervate them. There have been significant advances in the design and use of neuromodulation therapies currently in clinical trials or in use in the clinic to treat a variety of medical conditions affecting visceral organs. The goal of these therapies is to control end-organ function by use of electrical stimulation focused on the nerves that respond to and regulate the organ of interest. Although this approach shows great promise, it is also clear that a better understanding of the functional anatomy and connectome of the ANS would positively impact not only therapeutics development but would inform the underlying mechanisms of neural control of end organ function in both health and disease. To this end, we propose three specific aims focused on tool development, directed at establishing the connectome of the enteric nervous system (ENS) and the sympathetic and parasympathetic ganglia that innervate the gastrointestinal tract. The long-term goal is to better understand structure-function relationships in the ANS, which will inform development and use of next generation neuromodulation therapies. To this end we will develop new transgenic mice by intersectional targeting to direct Cre expression in three classes of enteric cholinergic neurons that define subclasses of neurons regulating enteric motor activity. We will generate pCAGGS-Brainbow 3.2 mice needed for determining functional neural circuits in the ANS. We will generate mice expressing optogenetic actuators and sensors that will be expressed in specific subsets of neurons in the ENS and sympathetic chain ganglia for functional neural network mapping using Ca2+ imaging and electrophysiology. We will use 3D-priniting technology to fabricate neural network models based on 3D analysis and segmentation of confocal images of enteric functional circuits and intestinofugal fibers. Lastly, we will develop tissue-clearing methods using modifications of the CLARITY technique in order to generate a 3D circuit and cellular phenotypic profiling (chemical coding) with high resolution.